LONG-TERM OBJECTIVES: The overall aim of this research is to understand the mechanisms involved in hemoglobin (Hb) switching - the developmental shifts in Hb types (embryonic to fetal/larval to adult Hbs) that occur in vertebrates of all classes, including humans. Elucidating these changes in gene expression in differentiating red blood cells (RBCs) will further the understanding of abnormal cellular differentiation that occurs in certain anemias and in carcinogenesis. Progress toward understanding this phenomena in a familiar animal model, the developing bullfrog Rana catesbeiana, is summarized in this proposal. SPECIFIC AIMS: 1. To test the hypothesis that during natural metamorphosis the newly differentiating erythroid cells transcribe and express only adult globin genes. 2. To test the hypothesis that when the pace of metamorphosis is retarded experimentally, larval as well as adult globin genes are expressed and are expressed in the same cells. 3. To test the hypotheses that both larval and adult Hbs are expressed in the same erythroid cells when severe anemia is induced in young adults, whereas embryonic and larval Hbs are expressed in separate cells when severe anemia is induced in premetamorphic tadpoles. 4. To test the hypotheses that induced retro-switching to embryonic Hb in tadpole liver co-cultured with tadpole kidneys requires close proximity of kidney and liver cells, whereas induced precocious switching to adult Hb in tadpole liver can be obtained by co-culture with factors isolated from culture medium conditioned by adult erythropoietic organs. 5. To test the hypothesis that thyroid hormones (which induce adult Hb in tadpole liver cultures) stimulate indirectly the growth and maturation of erythroid precursor cells whose program for Hb type has already been determined. METHODS: Means of manipulating Hb switching in vivo and in vitro (cell and organ cultures) are described. Gene expression will be assayed at the protein (globin and Hb) and mRNA levels using electrophoretic, immunochemical and nuclei acid hybridization techniques. Cell separations on Percoll gradients, cell cultures and colony-forming assays, and immunocytochemical procedures will be used to assess the cellular basis of Hb switching.